1. Field of the Invention
The invention relates generally to methods for preparing lactacystin and related compounds, to novel analogs of lactacystin and clasto-lactacystin xcex2-lactone, and their uses as proteasome inhibitors.
2. Description of Related Art
The Streptomyces metabolite lactacystin (1) inhibits cell cycle progression and induces neurite outgrowth in cultured neuroblastoma cells (Omura et al., J. Antibiotics 44:117 (1991); Omura et al., J. Antibiotics 44:113 (1991); Fenteany et al., Proc. Natl. Acad. Sci. (USA) 91:3358 (1994)). The cellular target mediating these effects is the 20S proteasome, the proteolytic core of the 26S proteasome, which is the central component of the ubiquitin-proteasome pathway for intracellular protein degradation. Mechanistic studies have established that lactacystin inhibits the proteasome through the intermediacy of the active species, clasto-lactacystin xcex2-lactone (2), which specifically acylates the N-terminal threonine residue of the proteasome X/MB1 subunit (Fenteany, et al., Science 268:726 (1995); Dick et al., J. Biol. Chem. 271:7273 (1996)). Lactacystin analogs are disclosed by Fenteany et al. (WO 96/32105). 
The ubiquitin-proteasome pathway is involved in a variety of important physiological processes (Goldberg et al., Chemistry and Biology 2:503 (1995); Ciechanover Cell 79:13 (1994); Deshaies, Trends Cell Biol.5:431 (1995)). In fact, the bulk of cellular proteins are hydrolyzed by this pathway. Protein substrates are first marked for degradation by covalent conjugation to multiple molecules of a small protein, ubiquitin. The resultant polyubiquitinated protein is then recognized and degraded by the 26S proteasome. Long recognized for its role in degradation of damaged or mutated intracellular proteins, this pathway is now also known to be responsible for selective degradation of various regulatory proteins. For example, orderly cell cycle progression requires the programmed ubiquitination and degradation of cyclins. The ubiquitin-proteasome pathway also mediates degradation of a number of other cell cycle regulatory proteins and tumor suppressor proteins (e.g., p21, p27, p53). Activation of the transcription factor NF-xcexaB, which plays a central role in the regulation of genes involved in the immune and inflammatory responses, is dependent upon ubiquitination and degradation of an inhibitory protein, IxcexaB-xcex1 (Palombella et al., WO 95/25533). In addition, the continual turnover of cellular proteins by the ubiquitin-proteasome pathway is essential to the processing of antigenic peptides for presentation on MHC class I molecules (Goldberg and Rock, WO 94/17816).
The interesting biological activities of lactacystin and clasto-lactacystin xcex2-lactone and the scarcity of the natural materials, as well as the challenging chemical structures of the molecules, have stimulated synthetic efforts directed toward lactacystin and related analogs. Corey and Reichard J. Am. Chem. Soc. 114:10677 (1992); Tetrahedron Lett. 34:6977 (1993)) achieved the first total synthesis of lactacystin, which proceeded in 15 steps and 10% overall yield. The key feature of the synthesis is a stereoselective aldol reaction of a cis-oxazolidine aldehyde derived from N-benzylserine to construct the C(6)-C(7) bond. In the synthesis reported by (Uno et al., J. Am. Chem. Soc. 116:2139 (1994)), stereoselective Mukaiyama-aldol reaction of a bicyclic oxazolidine silyl enol ether intermediate derived from, D-pyroglutamic acid is employed in C(5)-C(9) bond construction. This synthesis proceeds in 19 steps and 5% overall yield. Aldol reactions under basic conditions of a similar bicyclic oxazolidine intermediate form the basis of model studies reported by (Dikshit et al., Tetrahedron Lett. 36:6131 (1995)).
Aldol reactions of oxazoline-derived enolates feature prominently in the synthesis of lactacystin reported by Smith and coworkers (Suazuka et al, J. Am. Chem. Soc. 115:5302 (1993); Nagamitsu et al., J. Am. Chem. Soc. 118:3584 (1996)) and in the synthesis of (6R)-lactacystin reported by (Corey and Choi Tetrahedron Lett. 34:6969 (1993)); Choi Ph.D., Thesis, Harvard University, 44 (1995). In the former synthesis, which proceeds in 20 steps and 9% overall yield, the enolate is condensed with formaldehyde to install a single carbon atom, which must then be elaborated in a number of additional steps. In the Corey and Choi synthesis, the aldol reaction selectively provides the product of undesired stereochemistry, resulting in the eventual preparation of the C(6) epimer of lactacystin, which is devoid of biological activity.
Lactacystin has also been prepared in 22 steps and 2% overall yield from D-glucose (Chida et al., J. Chem. Soc., Chem. Commun. 793 (1995)). The biosynthetic pathway involved in production of the natural product has been investigated in feeding experiments involving 13C-enriched compounds (Nakagawa et al., Tetrahedron Lett. 35:5009 (1994)).
The reported syntheses of lactacystin are lengthy and proceed in low yield. Furthermore, none of these syntheses is readily adapted for analog synthesis. Thus, there is a need for improved methods for preparing lactacystin, clasto-lactacystin xcex2-lactone, and analogs thereof.
A first aspect of the present invention relates to a process for forming lactacystin or analogs thereof having Formula VI or clasto-lactacystin xcex2-lactone or analogs thereof having Formula VII: 
wherein
R1 is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;
R2 is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, or amido, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; and
R7 is alkyl, aryl, aralkyl, alkaryl, wherein any of said alkyl, aryl, aralkyl or alkaryl can be optionally substituted.
A second aspect of the present invention is directed to a method of forming formyl amides of Formula XIV: 
where R2 is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, or amido, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; and
R5 and R6 are independently one of alkyl or alkaryl; or R5 and R6 when taken together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclic ring, which may be optionally substituted, and which optionally may include an additional oxygen or nitrogen atom.
A third aspect of the present invention relates to forming tri-substituted oxazolines of Formula Ia or Ib: 
where R1 is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; and R4 is aryl or heteroaryl, either of which may be optionally substituted. The tri-substituted oxazolines of Formulae Ia and Ib are useful as starting materials in forming lactacysin, clasto-lactacystin xcex2-lactone or analogs thereof via the process described herein.
A fourth aspect of the present invention is directed to lactacysin, clasto-lactacystin xcex2-lactone or analogs of Formulae VI and VII that possess unexpected biological activity. Lactacystin, clasto-lactacystin xcex2-lactone, and analogs thereof possess biological activity as inhibitors of the proteasome. They can be used to treat conditions mediated directly by the function of the proteasome, such as muscle wasting, or mediated indirectly via proteins which are processed by the proteasome, such as the transcription factor NF-xcexaB.
A fifth aspect of the present invention relates to pharmaceutical compositions, comprising a compound of Formula VI or Formula VII, and a pharmaceutically acceptable carrier or diluent.
A sixth aspect of the present invention relates to methods of inhibiting proteasome function or treating a condition that is mediated directly or indirectly by the function of the proteasome, by administering a compound of Formula VI or Formula VII that possesses unexpectedly high activity in inhibiting the proteasome. Preferred Embodiments are directed to the use of a compound of Formulae VI or VII to prevent or reduce the size of infarct after vascular occlusion for example, for treating neuronal loss following stroke. An additional preferred embodiment is directed to the use of said compounds for treating asthma.
A seventh aspect of the invention relates to enantiomerically-enriched compositions of formyl amides of Formula XIV.
An eighth aspect of the present invention relates to novel individual intermediates, such as aldols of Formula II and aminodiols of Formula III: 
and individual steps within the multistep process for forming lactacystin, clasto-lactacystin xcex2-lactone or various analogs thereof.
A ninth aspect of the present invention relates to individual intermediates, such as compounds of Formulae XVII, XVIII and XIX: 
where X is a halogen, preferably Cl, Br or I, as well as individual steps within the multistep process for forming substituted oxazolines of Formula I.
Other features or advantages of the present invention will be apparent from the following detailed description, and also from the appending claims.